Contents

Journal Club Prep

Definitions

Epitope: Surface portion of an antigen capible of eliciting an immune response & of combining with the antibody produced to counter that response. [1]

Glycosylated: the adition of glycosyl groups to a protien to form a glycoprotien. [2]

Truncations: To shorten by or as if by cutting off, especially by cutting across at right angles to the long axis.[3]

Isomorphous replacement: A method of determining diffraction phases from the differences in intensity between corresponding reflections from two or more isomorphous crystals. Most commonly used in the determination of protein structures, where it is possible to derive isomorphous crystals of native protein and of heavy-atom derivatives. [4]

Porolate Ellipsoid: a figure generated by the revolution of an ellipse about its major axis. Contrasted with oblate spheroid. [5]

polymerization: a chemical reaction in which two or more molecules combine to form larger molecules that contain repeating structural units. Reduplication of parts in an organism. [8]

Trimeric complex: a polymer or a molecule of a polymer consisting of three identical monomers. [9]

lysis: the destruction or decomposition (of a cell)under influence of a specific agent. Mobilization of an organ by division of restraining adhesions. Gradual reduction of the symptoms of a disease.[10]

Non-neutralizing antibodies: produced after viral infection. They will bind to virus however they do not neutralize infectivity. On the contrary; they may enhance infectivity because antibodies can interact with receptors on macrophage.[11]

Neutralizing antibodies: They can fight off infection by neutralization. In which antibodies block virus infection. [12]

Lymphocytes: a type of white blood cell formed in lymphoid tissue [13]

SIV: Simian immunodeficiency viruses, SIV infects helper T cells. SIV-infected cells typically undergo apoptosis (programmed cell death) within one day of infection. It is similar to HIV and can be used as a model for HIV. However, the impact on human subjects is not like that of HIV. Affected Chimpanzies more than 32000 yrs ago. [14]

Outline

Introduction

Purpose: Give insight in the understanding HIV entry into cells.

For HIV to enter the cells a few things must happen:

sequential interaction of gp120 (viral exterior envelope glycoprotein) with CD4 glycoprotein and a chemokine receptor on the cell surface.

HIV can bypass the immune system

The structure, taken from x-ray crystal at 2.5A reslution, was taken of the HIV-1 gp120 core complex.

Cavity-laden interface of CD4-gp120 was seen

Chemokine reseptor was seen to have a conserved binding site

Conformation change when CD4 binds seems to occur

HIV-1, HIV-2 and SIV cause the distruction of CD4 lymphocytes in host, which can result in AIDS]

Viral envelop glycoprotiens

oligomeric/trimeric spikes on the surface of a virion.

attached to the viral membrane by gp41

Five variable regions (V1-V5) that were looked at in comparison to other primate immunodeficeiency viruses

The first four form surface exposed loops containing disulifed bonds at their bases.

gp120 regions are glycosylated

gp120 will bind to the domain that is closest to the N-terminal (CD4 domains)

Important CD4 binding sites have been determined (by mutagenesis)as well as conserved gp120 binding

Conformational change is induced when CD4 binds in the gp120 glycoprotein.

may expose/ form a binding site for chemokine receptors

CCR5 and CXCR4 (for HIV), these are necessary second receptors so that virus can enter

A crystallization strategy was designed for radical modification of the proteins surface. This was done because HIV glycoprotein 120’s properties of extensive glycosylation and conformational heterogeneity.

A theoretical analysis was done and was found that chances of crystal formation is increased by reducing surface heterogeneity and trials with multiple variants.

They then obtained crystals of gp 12- in the form of HXBc2 strain of HIV-1. They found that its capability of to interact with CD4 and other antibodies of relevance is preserved near wild type levels.

The structure in which they found the crystals to be was in ternary structure. The methods in find this consisted of molecular replacement, isomorphous replacement, along with density modification techniques.

Structure of gp120

prolate ellipsoid, overall profile is heat-shaped rather than circular

25 Beta-strands, 5 alpha helices and 10 defined loop segments.

Chemically determined disulphiede-bridge assignment

gp120s polypeptide chain; folded into two major domains

Inner domain: two-helix, two strand bundle with a five stranded beta sandwich at its terminiproximal end and a projection at the distal end

Outer Domain: stacked double barrel, lies along the side of the inner domain

Outer bundle and inner bundle axes are parallel

Barrels:

Proximal barrel of outer-domain:

Six stranded Beta sheets that is faced in mixed directions and twisted so that it interlocks with helix 2

Distal Barrel of stack:

seven stranded antiparellel beta barrel

Promimal and Distal Barrel share a hydrophobic core

Proximal End of outer domain:

Variable loops V4 and V5 and loops LD and LE…variable in sequences

Distal end of outer domain:

various aspects within this domain in turn hydrogen bonds with the V1/V2 stem that originate from the inner domain

Antiparallel, four stranded bridging sheet. Serves as a minidomain and is involved in ineraction of gp120 with CD4 and 17b antibody

body of outer domain as importance in CD4 binding due to its production of Beta 15 and helix alpha 3.

gp120 Structure as a whole:

didn’t find any similarity of the inner domain to known atomic structures. (There are missing terminal segments could prevent us from seeing this).

outer domain is has some similarities in certain portions. Superimposable fraction is limited for each of these segments.

sizeable amount conservation is seen

Variability and the amount of solvent exposure to residues are associated to each other. Specifically in relations with the conservation of the hydrophobic core.

All seven disulfide bonds in the core are conserved

Glycosylation sites are all exposed to the surface and are fairly conserved.

HIV variable segments are on loops are quite mobile.

Beta segments in outer domain arise from neutral mutation (rather than selective pressures) because they are on non-immmunogentic surfaces

CD4-gp12- interaction

CD4 in located in a depression located at the interface of the outer domain. The inner domain and bridging sheet at gp120 are also involved here.

Surface areas that are in contact are limited because. This is most likely due to a mismatch that creates large cavities that are occluded in the interface.

Phe 43 and Arg 59 of CD4 make multiple contacts centered on various residues. All conserved among primate immunodeficiency viruses.

Many gp120 residues which are covered by C4 show to be variable in sequences. This is partially due to a large interfacial cavity (water filled cavity).

Main-chain atoms of gp120 are the pathways for which gp120 residues can make contact with CD4

Interfacial Cavities

The solvent accessible surface of the ternary complex shows to contain cavities. The ones at which pg120 and CD4 are in contact are unusually large.

The larger cavity is lined by hydrophilic residues from both g120 and CD4. Will experience changes in side chain orientation, which in turn could cause it to be solvent accessible. Serves as a water buffer between gp120 and CD4. We see here that much variation can occur in required regions for CD4 binding. This may help the virus bypass antibodies.

Phe cavity is spherical. Located near the Phe43 of CD4 at the intersection of the inner domain, outer domain and bridging sheet. Deeply barried at hydrophobic interior of gp120. Only a few water molecules are seen here.

Another aspect that can affect the binding of antibodies associated with the CD4 binding site has to do with changes in cavity lining.

Antibody interface

17b antibody: neutralizing human monoclonal which was isolated from the blood of a subject with HIV. The contact surface is very acidic.

Well conserved among HIV-1 isolates

Chemokine-receptor site

CCR5 overlaps with 17b epitope at the site of interaction.

Induced by CD4 binding

Highly conserved residues

Oligomer and gp41 interaction

N and C termini of gp120 are VIP for ineraction with gp41 glycoprotien

gp41 will extended away from core and electornuetral surface is occluded in the oligomer interface.

This is also seen in influenza haemagglutinin

Conformational change in core gp120

CD4 binding induces a conformational change in gp120

Cavity lining residues have few structural restrictions, room for large substitutions into the cavity.

The pocket structure is closely connected to the briding sheet in the absence of CD4.

Phe43 cavity is in between the inner domain and the outer domain and the bridging sheet serving as a linchpin. Without this the structure could collapse.

Viral Evasion of Immune Responses

Most of protein surface is hidden b y glycosylation and oliogomeric occlusion from humoral immune responses. #*Neutralizing antibodies can only access the surface of (1) one in which overlaps with CD4 binding site and (2) one in which overlaps with chemokine-receptor binding site.

If CD4 is not bound the conformation may expose the underlying side chain variability

The binding pocket may also be a contributor to the escape

Conformational change may hide the epitote

Way in which contribute to antibody defenses may also help HUV avoid cellular immunity

Mechanistic implications for virus entry

gp120 is extremely important in the controlling and initiation of fusion. Fusion occurs between the viral membrane and the target cell membrane due to HIV surface proteins.

In order for entry one of the first steps is the binding of HIV-1 to the cellular receptor of CD$

Change in structure conformation is initiated by the binding of CD4 resulting in the creation of a mesastable oligomer.

Also, CD4 binding can cause the movment of the V3 loop and CD4i epitopes

CD4 binding stabilizes the bridging sheet and change the V3 region

Another aspect that causes conformational change is the binding of Chemokine receptors.

High affinity of interaction give reason to believe that CD4 and chemokine receptor remain bound to gp 120 during fusion.

Figure one

Shown is the overall structure which includes the gp120 protein (red), CD4 (yellow) and Fab7b antibody (dark and light blue).

The CDR3 loop is shown (dark blue) in which it contacts the gp120 protein.

extensive glycosylation and conformational heterogeneity caused the need for radical modification of the protein surface was devised to image it.

Figure Two

core is made up of 25 β-sheets, 5 α-helices, and 10 defined loop segments

polypeptide chain is folded into two main domains along with some digressions from this body

Briding Sheet lies mostly in the inner domain region but partially in the outer domain region serves as a mini domain